The production of heterologous proteins in bacterial hosts such as the bacterium Escherichia coli (hereinafter referred to collectively as E. coli and exemplified by E. coli except where otherwise expressly stated) is a powerful tool in the generation of many important biotechnological and medical products. This technique involves inserting the DNA encoding the product in question into an E. coli cell and using the cell to convert the genetic information into a functional protein.
Research over the past 20 years has demonstrated the ability of E. coli to serve as the expression host for a wide variety of proteins from numerous sources, ranging from other Gram-negative bacteria to mammalian proteins.
Improvements to the basic technology include the development of secretion mechanisms, whereby polypeptides are exported to the periplasmic space or the extracellular medium, as required for their folding and/or activity. The periplasmic space is of particular interest to the biotechnologist in terms of heterologous protein production in E. coli, due to its oxidising environment. This allows the formation of disulfide bonds, which are essential for correct folding and activity of many mammalian proteins of medicinal and/or biotechnological interest, such as antibody molecules.
Other improvements to fundamental expression systems in E. coli include greater control of expression of heterologous proteins, and novel peptide tags encoded on expressed molecules to facilitate their detection and purification.
Due to its extensive genetic and biochemical characterisation, E. coli is frequently the organism of choice for heterologous protein production experiments. E. coli also exhibits simple fermentation pathways and has a short doubling time, which is also advantageous. Furthermore, the nutritional (and sterility) requirements of E. coli are uncomplicated, relative to higher organisms.
A major disadvantage of E. coli as an expression host, however, is the fact that the yields attainable with this organism are relatively low, while it frequently also exhibits difficulties in synthesising proteins derived from eucaryotic sources. These difficulties can take the shape of an inability to carry out particular post-translational modifications of the translated polypeptide or, more fundamentally, an inability of the E. coli cellular machinery to fold the peptide in the first place. In instances such as the latter, the available solutions have been to translate the polypeptide in E. coli, followed by refolding in vitro—a time-consuming and highly inefficient process—or to switch expression host to a higher organism which can carry out the expression efficiently, but with the concomitant loss of advantages of E. coli, as outlined above.
While E. coli carries out the process of gene expression and protein production very efficiently with its own, natural proteins, it is considerably less productive when expressing proteins from other species. This is most likely due to an inability to correctly fold the translated polypeptide, or to successfully transport it to the appropriate subcellular compartment for assembly or folding. Such a deficiency may result from the E. coli “synthetic machinery” being unable to recognise or act upon heterologous proteins due to differences in such proteins relative to E. coli's native proteins. Alternatively, it may reflect an inability on the part of the host cells to express genes at the high levels demanded in such biotechnological experiments due to saturation of its normal gene expression and/or protein synthetic machinery. In such a scenario, the expressed protein typically forms large, insoluble aggregates consisting of multiple copies of the protein, which is non-functional and may be destroyed by the normal cellular machinery.
Furthermore, expression of heterologous genes in E. coli appears to frequently subject the cells to severe stress, leading to damage to the outer membrane of the host E. coli cell and leaking of the contents of the cell into the culture medium. This is typically followed by cell death via lysis of the E. coli cells.
This response of E. coli to expression of foreign genes has important implications for its potential in the production of a wide variety of heterologous proteins. With some foreign genes, E. coli has been found to be incapable of producing any functional protein; in cases in which E. coli folds the translated protein inefficiently or is overly stressed as a result of its expression, yields of the heterologous protein are dramatically reduced.
Researchers have attempted to overcome these problems with heterologous protein production in three main ways: i) genetic modification of the protein being expressed to improve its production in E. coli; ii) manipulation of the growth environment in order to reduce the stress on the expressing bacteria; and iii) co-expression in E. coli of natural folding-assisting molecules, termed chaperones, to improve production of the heterologous protein.
Genetic modification has proved successful with a number of proteins (Knappik, A. and Plückthun, A. (1995) Prot. Eng. 8:81-89; Wall, J. G. and Plïckthun, A. (1999) Prot. Eng. 12:605-611) but remains severely limited by the fact that solutions to expression problems that result from mutagenic modification are likely to be highly specific for the particular protein being expressed—whereas solutions that could be applied to all heterologous proteins expressed in E. coli would eliminate the need for labour-intensive, highly time consuming mutagenic studies to be repeated for each protein being produced.
Manipulation of the growth environment, for example, by modifying nutrients and temperature, has been shown to have a mildly positive effect in a number of cases, but would ultimately be expected to improve expression of reasonably efficiently expressed proteins rather than being able to overcome serious difficulties of expression or folding of specific proteins. The approach that appears to offer most hope in terms of a generally applicable solution is that of co-expressing folding assisting molecules that will enable the host E. coli cells to correctly express any or all heterologous proteins, without the need for further optimisation. To date, no single molecule has been identified, however, that improves the production of all heterologous proteins studied and, thus, again the difficulty arises of having to individually optimise expression for each heterologous protein.
Thus, a generally applicable solution to expressing traditionally “difficult” proteins in E. coli would make a highly significant contribution to the field of heterologous protein production.